Studies were undertaken to localize precisely the transcriptional enhancer elements within the long terminal repeats (LTRs) of different gibbon ape leukemia virus (GALV) strains and identify the cellular factor which binds to the enhancer region. 1) We have shown that the major element responsible for enhancer activity of the GALV LTR is contained within a 12 bp sequence of a 45 bp repeated DNA segment. 2) Using exonuclease II and DNAse I footprinting techniques we have identified a nuclear factor which binds specifically to the 123 bp enhancer sequence. It is present in cells which express GALV but not in cells which express GALV poorly. 3) We have demonstrated that the tumor promoter phorbol myristate acetate can enhance transcriptional activity of the GALV LTR 50-100 fold in various lymphoid cell lines. It also enhances expression of several other viral transcriptional elements including SV40, human T cell lymphotrophic virus type I, and Rous sarcoma virus, suggesting it works via a generalized mechanism. For practical purposes, PMA provides a convenient means for increasing transient expression of foreign DNA in lymphoid cells.